Abstract

The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13-Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.

Mcp1 is a mitochondrial PxP motif–containing Vps13 adaptor that interacts with Vps13 to…

Figure 6.

Mcp1 is a mitochondrial PxP motif–containing Vps13 adaptor that interacts with Vps13 to suppress ERMES mutants. (A) Schematic of Mcp1 highlighting a putative PxP interaction motif identified by FIMO by using a MEME motif generated from Ypt35 and Spo71 homologues. (B) Mutation of the motif (4–12Δ, P9,11A) blocked the ability of the Mcp1(1–58)-RFP-FYVE chimera to induce Vps13^GFP puncta in a ypt35 strain. (C) Quantitation of Vps13^GFP puncta per cell; n = 3, cells/strain/replicate ≥ 1,410. (D) The motif is required for coprecipitation of Vps13 and Mcp1. Vps13-GFP and WT and mutant forms of Mcp1-HA were overexpressed (OE) from the ADH1 promoter. IP, immunoprecipitation; WCL, whole-cell lysate. (E) Densitometry of D; n = 3. (F) The interaction between ADH1pr-driven overexpression of WT Mcp1 but not Mcp1 lacking the PxP motif recruits Vps13^GFP to mitochondria marked by preCox4-RFP (red). (G) Loss of the Mcp1 PxP motif is synthetic lethal with mdm10Δ. (H) Overexpression of WT but not mutant Mcp1suppresses the mitochondrial morphology defect of a strain lacking the ERMES subunit Mdm10. In contrast, loss of YPT35 does not rescue the mdm10Δ mitochondrial morphology defect with or without expression of VPS13 from two copies of the gene, nor does it block rescue by the dominant suppressor Vps13(D716H). DAPI was used as a mitochondrial marker. Bars, 2 µm. Error bars indicate SEM.

Model of Vps13 recruitment by adaptor proteins with PxP motifs. Ypt35, Spo71, and Mcp1 all contain PxP motifs that compete to recruit Vps13 to endosomes/vacuole/NVJ, the prospore membrane, and mitochondria respectively. Each adaptor shows PxP-dependent binding to a central repeat region of Vps13 that overlaps the DUF1162 domain; we propose this domain be named the VPS13 adaptor binding (VAB) domain.